The de novo Due Ferri single chain (DFsc) protein contains a binuclear active site comprised of two histidine and four glutamate residues. Originally designed to mimic non-heme diiron enzymes, we have explored the ability of the DFsc to serve as a scaffold to model the structure and reactivity of coupled binuclear copper enzymes. To this end, we have created a library of DFsc variants with systematic glutamate to histidine mutations, ranging from the original two histidine configuration to a six histidine configuration. The produced and purified proteins all appear to coordinate between one and two copper ions. Circular dichroism spectroscopy indicates that most contain the expected alpha-helical fold, with the four histidine variant particularly well-folded even in the absence of metal ions. All of the proteins appear capable of activating molecular oxygen and performing simple two-electron oxidation reactions. The pH dependence of the reaction rates has been explored. From these data, we can begin to understand how the active site composition tunes the metal binding and catalytic properties of dicopper enzymes.

Systematic evaluation of copper binding and activation by de novo Due Ferri single chain proteins

2019 Spring National Meeting

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